The present invention relates in part to methods of using tyramide in order to enhance the detection of analytes by flow cytometry, preferably using catalyzed reporter deposition and amplification staining. In preferred embodiments, the analytes detected according to the instant methods are cellular antigens, which may be intracellular or expressed on the surface of cells. The invention also relates in part to methods and compositions for use in amplification staining of analytes.
The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Flow cytometry is a sensitive and quantitative method for measuring the fluorescence or light scatter of particles or cells. This method has been widely used to study cellular physiology, especially as it relates to the immune system and control of the cell cycle. Nolan et al, xe2x80x9cThe Emergence of Flow Cytometry for Sensitive, Real-time Measurements of Molecular Interactionsxe2x80x9d, Nature Biotechnology, Vol. 16, (1998), which is incorporated by reference herein in its entirety including any drawings, describe recent flow cytometry developments for fields as diverse as ligand binding and enzyme kinetics, drug screening, diagnostics and detection of soluble agents, and DNA sequence detection or analysis. They describe developments such as advances in automated sample handling, molecular approaches for incorporating affinity tags or fluorescent probes into proteins and the availability of microsphere reagents that enable multiplexing.
Flow cytometric analysis of cellular antigens is a technology used in both medical diagnostic laboratories and biomedical research laboratories. In clinical practice flow cytometry is used, e.g., for samples derived from patients infected with human immunodeficiency virus type 1, patients with leukemias and lymphomas, and patients with primary immunodeficiences.
Various methods have been described for assaying biological samples with amplified reporter systems. Bobrow et al., U.S. Pat. No. 5,196,306, U.S. Pat. No. 5,583,001 and U.S. Pat. No. 5,731,158, which are all herein incorporated by reference in their totality including any drawings, describe methods for detecting or quantitating analytes using an analyte dependent enzyme activation system as well as catalyzed reporter deposition methods. Specifically, Bobrow et al. describe colorimetric and fluorometric solid phase enzyme immunoassays which are enhanced by amplification of the reporter molecules.
Chao et al., xe2x80x9cImmunofluorescence Signal Amplification By The Enzyme-Catalyzed Deposition Of A Fluorescent Reporter Substrate (CARD)xe2x80x9d, Cytometry 23:48-53 (1996), describe a CARD system that uses horseradish peroxidase substrate Cy3.29-tyramide to deposit fluorogen molecules onto fixed tissues and cells as well as proteins bound to nitrocellulose membranes, with up to a 15 fold increase over standard indirect immunofluorescence methods.
Malisius et al., xe2x80x9cConstant Detection of CD2, CD3, CD4, And CD5 In Fixed and Paraffin-Embedded Tissue Using The Peroxidase-Mediated Deposition Of Biotin-Tyramidexe2x80x9d, The Journal of Histochemistry and Cytochemistry, Vol. 45(12):1665-1672, (1997), describe a method for enhancing detection of leukocyte antigens in formalin-fixed tissue samples.
Lollini et al., xe2x80x9cFlow Cytometry on Intracellular Antigens After Tyramide Signal Amplification,xe2x80x9d Immunological Blackboard: Bulletin of the Gruppo Di Cooperazione in Immunologia, Vol. 1, Number 2 (1998), which is incorporated herein by reference in its entirety, including any drawings, describes tyramide signal amplification (TSA) for detection of intracellular antigens by flow cytometry. Lollini et al. indicates that TSA is not superior to conventional techniques for detecting surface antigens on live cells, stating for example on page 5, xe2x80x9c(t)he main problem appeared to be a high level of spontaneous activation and non-specific binding of the fluorescent substrate to live cell membranes.xe2x80x9d
Karkmann et al., xe2x80x9cEnzymatic Signal Amplification For Sensitive Detection Of Intracellular Antigens By Flow Cytometry,xe2x80x9d J. Immunol. Meth. 230:113-120 (1999), which is incorporated herein by reference in its entirety, including any drawings, describes TSA for detection of intracellular cytokines by flow cytometry. The Karkmann et al. reference states that direct detection of fluorochrome-labeled tyramide is problematic, xe2x80x9cprobably due to hydrophobic interactions with the cell membranexe2x80x9d Thus, the Karkmann et al. reference uses a complicated indirect staining method, in which antibody selective for an antigen of interest, coupled to peroxidase, catalyzes the deposition of biotin-tyramide, which is detected by fluorescently labeled streptavidin.
Despite progress towards catalyzed reporter deposition methods for use in detecting analytes, there remains a great need in the art for materials and methods that provide enhanced detection of analytes by enzymatic signal amplification, particularly for use in flow cytometry.
The instant invention features materials and methods for enhancing the detection and/or quantitation of an analyte of interest by flow cytometric and immunostaining analysis. In preferred embodiments, an analyte is a cellular antigen, which can be either intracellular or expressed on the surface of cells.
The invention provides methods for catalyzed reporter deposition staining of intracellular antigens that are simple, and that provide signal amplification levels of 10-fold or more in comparison to standard flow cytometry methods. Moreover, the invention also provides a method for tyramide coating cells for flow cytometry, wherein cells are preferably exposed to a catalyzed reporter deposition system which results in specific tyramide coating of cells which contain or express an analyte of interest. Additionally, the invention further provides materials and methods for improving catalyzed reporter deposition staining of antigens generally, including the staining of fixed sections, fixed and permeabilized cells, particles, and live cells.
Thus, in a first aspect, the present invention discloses materials and methods that provide an enhanced signal for detection of an intracellular analyte of interest in a cell sample by flow cytometric methods. The methods disclosed comprise the steps of fixing the cells in the cell sample, permeabilizing the cells, and catalyzing the deposition of tyramide in those cells that comprise the analyte of interest. The present invention allows for detection of analytes which are present in low copy number in the cell sample by increasing the signal from an analyte of interest from 5-fold to 50-fold or more in comparison to standard flow cytometric methods.
The terms xe2x80x9cfixxe2x80x9d and xe2x80x9cfixingxe2x80x9d as used herein with regard to cells, refers to various chemical and physical methods well known to the skilled artisan that render the contents of a cell insoluble. Preferred fixation methods, which generally rely on crosslinking and/or rapid dehydration, can include the use of one or more chemicals such as formaldehyde, paraformaldehyde, glutaraldehyde, acetic acid, methanol, ethanol, and acetone.
The term xe2x80x9cpermeabilizexe2x80x9d as used herein with regard to cells, refers to various methods that allow an increased amount of one or more agents to pass into the interior of a cell. The skilled artisan will understand that a cell is ordinarily surrounded by a semipermeable plasma membrane. Permeabilizing agents can act to open the plasma membrane, allowing molecules to enter the cell and to reach a concentration within the cell that is greater than that which would ordinarily be attained. Permeabilizing can also allow molecules to enter a cell that would ordinarily not enter the cell, for example due to a large molecular size or because the molecule is highly charged. Preferred permeabilizing agents are detergents such as CHAPS, cholic acid, deoxycholic acid, digitonin, n-dodecyl-xcex2-D-maltoside, glycodeoxycholic acid, n-lauroylsarcosine, lauryl sulfate, saponin, and triton X-100; and organic alcohols such as methanol and ethanol. These examples are not meant to be limiting, and the skilled artisan will know various additional methods to render cells permeable, such as mechanically disrupting the cell membrane by scraping cells that have adhered to a solid surface.
The term xe2x80x9ccatalyzing the deposition of tyramidexe2x80x9d refers to any process which results in the enzymatic deposition of tyramide in or on the surface of cells, or any other particle such as a cell organelle, liposome, latex particle, magnetic bead, etc., comprising an analyte of interest. It is believed that, in the presence of oxygen radicals, short lived tyramide radicals are formed which form covalent linkages with aromatic molecules such as certain amino acids (tyrosine and tryptophan for example) found in most proteins. Oxygen radicals are preferably formed by the catalytic activity of an enzyme such as a peroxidase in the presence of an appropriate substrate. Catalyzed deposition methods have the ability to amplify the intensity of a signal obtainable from an analyte of interest, due to the deposition of plurality of tyramide molecules at or near to a molecule of analyte.
In particularly preferred embodiments, a binding partner that specifically binds to an analyte of interest, such as an antibody or a fragment thereof (e.g. Fab, (Fab)2, Fabxe2x80x2, (Fabxe2x80x2)2) directs the deposition of tyramide. In these embodiments, the binding partner is covalently or noncovalently conjugated to an enzyme that produces oxygen radicals. Upon the addition of tyramide and a suitable enzyme substrate, it is believed that tyramide radicals are formed that bind to cell surfaces or interior structures to which the radical is in closest proximity. In particularly preferred embodiments, the tyramide is covalently or noncovalently conjugated to a signal generating element.
The skilled artisan will understand that the binding partner need not be directly conjugated to the enzyme, but can be indirectly linked via one or more additional molecules such as a second binding partner that specifically binds to the first binding partner. For example, a binding partner that specifically binds to an analyte of interest can itself be bound by a second antibody that is conjugated to an enzyme. In other embodiments, the binding partner may be conjugated to a molecule such as biotin; biotin can then be bound by a second binding partner such as avidin or streptavidin that is conjugated to an enzyme. Similarly, the binding partner may be conjugated to a molecule, for example a hapten such as FITC, digoxigenin, trinitophenol (TNP), or dinitrophenol (DNP), which can then be bound by a second antibody directed against the hapten, with the second antibody conjugated to the enzyme. The skilled artisan will recognize that numerous other methods can be used to indirectly link the binding partner to the enzyme.
In particularly preferred embodiments, a binding partner is an antibody fragment, such as an Fab, (Fab)2, Fabxe2x80x2, (Fabxe2x80x2)2, that is conjugated to an enzyme, for example horseradish peroxidase.
The skilled artisan will also understand that tyramide also need not be directly conjugated to a signal development element. In certain embodiments, tyramide may be indirectly bound to a signal developing element via a second molecule, such as a hapten bound to tyramide which is specifically bound by an anti-hapten antibody that has been conjugated to a signal development element. In other embodiments, tyramide may be conjugated to a molecule such as biotin; biotin can then be bound by a second binding partner such as avidin or streptavidin that is conjugated to a signal development element.
The term xe2x80x9ccellsxe2x80x9d as used here refers to the smallest unit of living structure capable of either aided or unaided existence, composed of a membrane-enclosed interior which may contain a nucleus or nucleoid, free compact DNA, and/or other organelles such as mitochondria, the golgi apparatus, centrioles, endoplasmic reticulum, vacuoles, microsomes, lysosomes, ribosomes and the like. The cells can be bacterial cells as well as eukaryotic cells such as plant cells, yeast or fungal cells or mammalian cells. In a preferred embodiment, the live cells are mammalian cells. Cell types can include but are not limited to basal cells, epithelial cells, erythrocytes, platelets, lymphocyte, T-cells, B-cells, natural killer cells, granulocytes, monocytes, mast cells, Jurkat cells, neurocytes, neuroblasts, cytomegalic cells, dendritic cells, macrophages, blastomeres, endothelial cells, HeLa cells, tumor cells, interstitial cells, Kupffer cells, Langerhans cells, littoral cells, tissue cells such as muscle cells, adipose cells, CHO cells, KFL9 cells, K562 cells, enucleated cells and the like as well as cells readily prepared and sold by immunological and microbiological resources currently. In certain embodiments, cells are grown as cell cultures. In particularly preferred embodiments, the cells are obtained from a patient.
The term xe2x80x9cculturedxe2x80x9d as used herein in reference to cells can refer to one or more cells that are undergoing cell division or not undergoing cell division in an in vitro environment. An in vitro environment can be any medium known in the art that is suitable for maintaining cells in vitro, such as suitable liquid media or agar, for example. Specific examples of suitable in vitro environments for cell cultures are described in Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.; Cells: a laboratory manual (vol. 1), 1998, D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold Spring Harbor Laboratory Press; and Animal Cells: culture and media, 1994, D. C. Darling, and S. J. Morgan, John Wiley and Sons, Ltd., each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings. Cells may be cultured in suspension and/or in monolayers with one or more substantially similar cells. Cells may be cultured in suspension and/or in monolayers with a heterogeneous population of cells. The term xe2x80x9cheterogeneousxe2x80x9d as utilized in the previous sentence can relate to any cell characteristics, such as cell type and cell cycle stage, for example. Cells may be cultured in suspension, cultured as monolayers attached to a solid support, and/or cultured on a layer of feeder cells, for example.
The term xe2x80x9cpatientxe2x80x9d as used herein refers to an individual seeking medical diagnosis or treatment. A patient may be an animal, for example in a veterinary setting, but most preferably is a human.
By xe2x80x9caided existencexe2x80x9d is meant adding components to the buffer or medium containing the cells which allows the cell to remain viable.
The term xe2x80x9clow copy numberxe2x80x9d means that the analyte of interest is present on or in the cell but is not represented in an easily detectable amount. An aspect of the present invention is that rare, hard to detect analytes may be readily detected by the amplified signal intensity obtainable from the cell caused by the amplification of the labeling molecule. Hence, a low copy number analyte, such as the Fas ligand, would not have to be over-expressed in order to be detected by flow cytometry. The low copy number is preferably less than 20,000 molecules/cell, more preferably less than 10,000 molecules/cell and most preferably less than 2,000 molecules/cell.
The term xe2x80x9cbinding partnerxe2x80x9d as used herein refers to biochemical or chemical molecules such as polypeptides, glycoproteins, glycolipids, lipids, or nucleic acids which bind to the analyte of interest or to a first binding partner which specifically binds to the analyte of interest. Binding partners may be attached naturally through contacting a molecule with a receptor for such a molecule. The polypeptides can be conjugated proteins, antibodies and the like. Hence, a binding partner may consist of an antibody bound to a label or an enzyme bound to a binding partner, or an antibody bound to a binding partner. Pairs of binding partners can be but are not limited to, (i) streptavidin and biotin, (ii) an antibody or antibody fragment and an epitope, (iii) an antibody or antibody fragment and a protein, (iv) a protein and a receptor molecule or receptor protein, (v) a nucleic acid and a nucleic acid, (vi) a nucleic acid and a protein, (vii) a hormone and a hormone receptor, (viii) a cytokine and a cytokine receptor. The nucleic acids can be DNA, RNA, cDNA, mixed oligonucleotides, peptide nucleic acids (PNA), Locked Nucleic Acids (LNA) as described in Koshkin, et al., Tetrahedron Letters 1998 39:4381-4384, which is incorporated herein by reference in its entirety including any drawings, and the like. In a preferred embodiment the binding partner with specificity to a first binding partner which has bound the cellular analyte of interest, has enzymatic activity. It would be clear to one of skill in the art that various combinations of binding partners which are capable of binding by either covalent or non-covalent means can be used in the invention to catalyze the deposition of tyramide in an analyte-dependent fashion.
The term xe2x80x9ccontactingxe2x80x9d as used herein refers to bringing the cells into close proximity with a molecule in a manner which allows the cells to interact with the moelcule. xe2x80x9cContactingxe2x80x9d can also preferably refer to bringing cells into close proximity with a binding partners in a manner which allows the binding partners to interact with its conjugate partner and thereby bind. xe2x80x9cContactingxe2x80x9d may also refer to bringing cells into close proximity with an enzyme substrate in a manner which allows any previously bound partners which posses enzymatic activity to interact with the substrate.
The term xe2x80x9canalyte of interestxe2x80x9d as used herein refers to a molecule that is to be detected by the methods described herein. The molecule can be a protein, glycoprotein, glycolipid, lipid, a nucleic acid, or a biochemical or chemical molecule as defined above. An analyte of interest can be an analyte which changes in abundance in a cell in a pathological state; in these embodiments, a signal obtained from the methods described herein can be correlated to the diagnosis, status, or prognosis of a disease. In other preferred embodiments, an analyte of interest is not a natural component of a cell, but has been expressed in the cell by recombinant DNA techniques well known to the skilled artisan.
In certain embodiments, the analyte of interest is an intracellular antigen such as intracellular cytokines, antigens (e.g., viral antigens, nuclear antigens, cytoplasmic antigens, organellar antigens), enzymes, cytoskeletal molecules, glycolipids, lipids, glycans, chaperones, RNA, DNA, messenger RNA, and ribosomal RNA, signal transduction proteins, and structural proteins.
In other embodiments the analyte of interest is a cell surface expressed or intracellular molecule such as, but not limited to, cell surface ligands such as Fas ligand (which binds CD95) and the ligands for CD1 through CD247, CD1 through CD166 as disclosed in xe2x80x9cLeukocyte Typing VI: White Cell Differentiation Antigensxe2x80x9d Edited by Kishimoto et al., Garland Publishing, Inc. New York 1997, which is incorporated herein by reference in its entirety including any drawings, additional CDs, such as those published in the Proceedings of the 7th International Workshop and Conference on Human Leucocyte Differentiation Antigens, Harrogate, Jun. 20-24 2000, Oxford University Press (also available on the internet at http://gryphon.jr2.ox.ac.uk/cdlist.htm), hormone receptor molecules, cytokine receptor molecules, MHC class I, MHC class II, cell receptors for IgG, and IgE, cell receptors for complement components such as receptors for C3a, C5a, CR1 and CR3, T-Cell or B-Cell receptor molecules, viral antigens, tumor antigens, histocompatibility antigens, differentiation antigens, T-cell antigen, Ly antigen, Ly-6 (Classon et al., Dev. Immunol. Vol. 6(1-2):149-156, 1998, Kato et al., Otolarvngol Head Neck Surg. Vol. 119(4):408-411, 1998, IgD, IgM and the like. Also included are cell surface and intracellular molecules within families of molecules such as those disclosed above.
The term xe2x80x9cenzymatic activityxe2x80x9d as used herein refers to the ability of the binding partner to act as a catalyst to induce chemical changes in other substances. In one embodiment the enzymatic activity catalyzes the dehydrogenation (oxidation) of various substances in the presence of hydrogen peroxide. In a preferred embodiment the enzymatic activity refers to the reaction between a peroxidase directly or indirectly bound to a binding partner and a peroxide substrate. The enzymatic activity could also be the result of the reaction between enzymes such as, but not limited to, hydrolases, oxidases, phosphatases, esterases and glycosidases and their respective substrates. Most preferably, the enzymatic activity is horseradish peroxidase.
The terms xe2x80x9cdetectable labelxe2x80x9d and xe2x80x9csignal generating elementxe2x80x9d as used herein refer to substances or molecules which generate a signal that can be detected by a detection device, such as by flow cytometric analysis. Detectable labels are preferably fluorochromes including, but not limited to, fluorescein, phycoerythrin, CY3, CY5, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-CY5, and the like. These examples are not meant to be limiting. Other preferred labels are molecules that produce a visible color or that are chemiluminescent. Numerous suitable detectable labels are known to the skilled artisan.
The term xe2x80x9cfluorochromexe2x80x9d as used herein refers to a molecule that absorbs a quantum of electromagnetic radiation at one wavelength, and emits one or more photons at a different, typically longer, wavelength in response. In particularly preferred embodiments, a fluorochrome can be a member of a pair of fluororchromes that exhibit fluorescence energy transfer. In such a pair, absorbance of a quantum of electromagnetic radiation at one wavelength by the donor fluorochrome of the pair results in emission that is absorbed by the acceptor fluorochrome of the pair, resulting in emission by the acceptor fluorochrome.
The term xe2x80x9cflow cytometryxe2x80x9d as used herein refers to methods for analysis of cell or particulate samples well known to the skilled artisan, such as those provided by Becton-Dickinson, Cytomation, Partec, Luminex, or Beckman-Coulter. Flow cytometry can encompass multiparametric DNA analysis, platelet studies, reticulocyte enumeration, cell biology/functional studies, innovative research in immunobiology, cell physiology, molecular biology, genetics, microbiology, water quality and plant cell analysis as well as a broad range of research applications. Current flow cytometers are manufactured with the ability to measure more than one, preferably four or more separate detectable labels simultaneously. Using methods for flow cytometric analysis, a specific labeled molecule, such as an antibody, is added to the cellular or particulate sample believed to contain an analyte of interest. The antibody is labeled with an appropriate detectable label, such as a fluorochrome, which permits detection of those cells or particles comprising the analyte of interest at a detectable level. The analysis can involve quantitation and/ or detection of the analyte, and may also involve sorting or harvesting the cells or particles possessing the analyte of interest.
The terms xe2x80x9cstandard flow cytometryxe2x80x9d and xe2x80x9cstandard staining for flow cytometryxe2x80x9d as used herein refer to flow cytometry methods in which cells or particles are stained for an analyte of interest using procedures that do not involve catalyzed deposition of a reporter molecule such as tyramide. Standard flow cytometry includes direct staining of an analyte of interest using one or more labeled molecules attached to a binding partner for the analyte. Standard flow cytometry also includes non-catalysis-based amplification methods, in which, for example, multiple layers of binding pairs are used to produce an increased number of sites at which a labeled molecule may be bound.
The term xe2x80x9cfold enhancementxe2x80x9d as used herein in reference to the amplification of signal obtained by catalyzed deposition procedures refers to methods that compare two different samples or staining techniques by flow cytometry. The fold enhancement is measured by comparing the peak or mean signals obtained from the two comparison groups, as measured by channel separation in the flow cytometer. The skilled artisan will understand that the fold enhancement requires discrete peaks in order to be properly measured. Furthermore, proper isotype and subtype matched antibodies must be used in order to obtain proper control measurements. The samples, and the appropriate controls, must all be run using identical settings (e.g., voltage) on the flow cytometer, and the signal histograms obtained from each sample and control must be similar in shape in order to properly measure the fold enhancement. Moreover, the mean channel numbers of the isotype controls must be approximately equal in order to properly compare two different staining results. If they are not approximately equal, results from two staining procedures can still be compared by using the same number to multiply or divide the mean channel numbers of the histograms representing the cells stained with both the control immunoglobulin and the specific immunoglobulin of one staining procedure in order to make the mean channel numbers of the histograms representing the cells stained with the control immunoglobulin similar or identical for both staining procedures being compared. Alternatively, the signal histograms can be altered by changing the instrument settings and re-running the samples. Samples of cells stained with the control antibody and with the specific antibody by one procedure can be re-run at new voltage settings so that the peak or mean channel number for the control is similar to the peak or mean channel number for the control of cells treated by a different procedure. These 2 methods of comparison give similar results.
In preferred embodiments, the methods for detecting the presence of analytes described herein provide between a 5- and 100-fold, or greater, enhancement in comparison to standard flow cytometry methods, when proper isotype and subtype matched antibodies are used to obtain proper control measurements. Most preferably, the method provides a 5-fold enhancement, a 10-fold enhancement, a 15-fold enhancement, a 20-fold enhancement, a 25-fold enhancement, a 30-fold enhancement, a 40-fold enhancement, a 50-fold enhancement, a 60-fold enhancement, a 75-fold enhancement, a 100-fold enhancement, a 200-fold enhancement, a 500-fold enhancement, and a 1000-fold enhancement in comparison to standard flow cytometry methods, as the term xe2x80x9cfold enhancementxe2x80x9d is defined herein.
In certain embodiments, the deposition of tyramide is catalyzed by (i) incubating the fixed and permeabilized cells with a binding partner that specifically binds to an intracellular analyte of interest and that is conjugated to an enzyme capable of catalyzing the deposition of tyramide; (ii) removing unbound binding partner from the cells; and (iii) contacting the bound binding partner with tyramide, whereby the enzyme catalyzes the deposition of tyramide in those cells comprising the intracellular analyte. The skilled artisan will understand that unbound binding partner can be removed by a number of methods known in the art, such as washing the cells.
In preferred embodiments, one or more binding partners are incubated with fixed and permeabilized cells in a medium comprising serum. The medium preferably comprises from about 10% to about 100% serum, measured w/v or v/v. For example, 1 mg of a lyophylized antibody may be dissolved in serum to a total volume of 100 mL, resulting in the antibody being dissolved in a medium comprising 99% serum w/v. Similarly, 1 mL of antibody dissolved in water may be diluted to a total of 100 mL with serum, resulting in the antibody being dissolved in a medium comprising 99% serum v/v. In particularly preferred embodiments, the medium comprises at least about 10% serum, 20% serum, 30% serum, 40% serum, 50% serum, 60% serum, 70% serum, 80% serum, 90% serum, 95% serum, 99% serum, and 100% serum. The percentages are measured by volume, and the term xe2x80x9caboutxe2x80x9d in this context refers to +/xe2x88x920.5%. A preferred serum is fetal bovine serum.
In other preferred embodiments, one or more binding partners are incubated with fixed and permeabilized cells in a medium that also comprises a detergent, such as CHAPS, cholic acid, deoxycholic acid, digitonin, n-dodecyl-xcex2-D-maltoside, glycodeoxycholic acid, n-lauroylsarcosine, lauryl sulfate, saponin, triton X-100, at a concentration of from 0.1% to 25%. Most preferably, the medium comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, and 25% detergent. The percentages are measured by volume for those detergents that are a liquid, and by weight for those detergents that are solids, and the term xe2x80x9caboutxe2x80x9d in this context refers to +/xe2x88x920.05%. Particularly preferred is a medium comprising at least about 95% serum and about 0.2% saponin.
In other preferred embodiments, the enzyme that catalyzes the deposition of tyramide is contacted with tyramide in an amplification medium that has a low ionic strength and/or comprises one or more aprotic solvents. As discussed above, the skilled artisan will understand that the amplification medium may also comprise a substrate for use by the enzyme in producing oxygen radicals necessary for tyramide deposition.
The term xe2x80x9clow ionic strengthxe2x80x9d as used herein relates to both the concentration of ions in a solution, and the charge of those ions. The ionic strength of a solution is given by the formula (xcexa3cizi2)/2, where ci is the concentration of an ion in solution, and Zi is the charge of the ion. A low ionic strength preferably refers to an ionic strength equal to or less than that of a 0.2 M sodium borate solution, pH 7 to 9; more preferred low ionic strengths are equal to or less than 0.1 M, 0.05 M, 0.02 M, 0.01 M, 0.005, 0.001, and 0.0001 M, having a pH of 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, and 9; and a most preferred low ionic strength is that of deionized water.
The term xe2x80x9caboutxe2x80x9d in the context of concentration refers to +/xe2x88x925%, and in the context of pH refers to +/xe2x88x920.1 pH units.
The term xe2x80x9caprotic solventxe2x80x9d as used herein refers to polar organic solvents that lack a hydrogen bond donor group. Some examples of aprotic solvents are acetone, dimethyl sulfoxide, acetonitrile, hexamethyl phosphoramide, n-methyl pyrrolidine, and dimethyl formamide. A preferred medium comprising an aprotic solvent is an aqueous medium comprising from about 1% to about 25% aprotic solvent. Most preferably, the medium comprises about 20% aprotic solvent, about 15% aprotic solvent, about 10% aprotic solvent, about 7.5% aprotic solvent, about 5% aprotic solvent, about 2.5% aprotic solvent, and about 1% aprotic solvent. The percentages are measured by volume, and the term xe2x80x9caboutxe2x80x9d in this context refers to +/xe2x88x920.5%.
In other preferred embodiments, the enzyme that catalyzes the deposition of tyramide is contacted with tyramide in an amplification medium comprising amino acids, dipeptides and/or oligopeptides as the buffering agent. Preferred amplification media comprise, for example, glycylglycine at a concentration of between 0.2 M to 0.0001 M and at a pH of 7 to 9. Most preferred are glycylglycine buffers of 0.1 M, 0.05 M, 0.05 M, 0.02 M, 0.01 M, 0.005, 0.001, and 0.0001 M, having a pH of 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, and 9. The amplification medium can also comprise one or more salts, such as sodium chloride, potassium chloride, magnesium chloride, etc., at a concentration of from 3 M to 0.001 M, and/or one or more aprotic solvents. In preferred embodiments, the salt(s) are at a concentration of 2.5 M, 2, M, 1.5 M, 1.25 M, 1 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.45 M, 0.4 M, 0.35 M, 0.3 M, 0.25 M, 0.2 M, 0.15 M, 0.1 M, and 0.05 M. In a particularly preferred embodiment, the amplification medium comprises 0.02 M glycylglycine, pH 8.0, and 1 M NaCl.
In principle, any buffering agent can be used in an amplification medium depending on the desired pH of the final medium. Preferred amplification media have a pH of between 7 and 9. Preferred buffers are ACES, ADA, BES, bicine, bis-tris, CAPS, CHES, diethylmalonate, glycylglycine, glycinamide HCl, HEPES, HEPPS, imidazole, MES, MOPS, PIPES, POPSO, TAPSO, TES, tricine, tris, bicarbonate, and borate. Preferred buffering agent concentrations are less than 2 M; most preferred concentrations are 0.2 M, 0.1 M, 0.05 M, 0.05 M, 0.02 M, 0.01 M, 0.005, 0.001, and 0.0001 M, having a pH of 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, and 9.
When a buffering agent comprises amino acids, dipeptides and oligopeptides, the amino acid residues making up the buffering agent can be either purified L or D stereoisomers, or a mixture of both L and D stereoisomers. The skilled artisan will understand, however, that certain amino acids (e.g., glycine) are non-chiral. Particularly preferred are buffering agents comprising one or more amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, hydroxyproline, hydroxylysine, desmosine, isodesmosine, xcex2-alanine, homocysteine, homoserine, citrulline, omithine, and xcex3-aminobutyric acid. Most preferred are buffering agents comprising at least one glycine residue.
In another aspect, the present invention features a method for tyramide labeling cells for multiparameter flow cytometric analysis by contacting the cells and/or particles with the following; a first binding partner specific for a first analyte of interest, a second binding partner with enzymatic activity and which specifically binds to the first binding partner, a substrate for the enzymatic activity of the second binding partner, and a labeling molecule containing tyramide. After tyramide deposition and the addition of a detectable marker, the enzymatic activity is inhibited. The cells are then contacted with a third binding partner specific for a second analyte of interest, a fourth binding partner with enzymatic activity and which specifically binds to the third binding partner, a substrate for the enzymatic activity of the fourth binding partner, and a labeling molecule containing tyramide and specific for the third or fourth binding partners.
The skilled artisan will understand that, in performing double labeling methods, the first (or third) binding partner described above may itself be covalently or noncovalently conjugated to an enzyme that produces oxygen radicals. In such a case, the second (or fourth) binding partner would not be required. Alternatively, the first (or third) binding partner may be bound to a molecule such as FITC, digoxigenin, or dinitrophenol (DNP), which can then be bound by a second (or fourth) binding partner directed to FITC, digoxigenin or DNP, and which is conjugated to the enzyme. The skilled artisan will recognize that numerous other methods can be used to indirectly link a binding partner to the enzyme.
Tyramide is deposited in or on the cells comprising the analytes of interest as a result of the enzymatic activities of the second and fourth binding partners. Detectable markers can be added after tyramide deposition to facilitate flow cytometric analysis. The first and second detectable markers can be the same fluorochrome molecule which is attached to a binding partner specific for the tyramide containing molecules and would be detected by an increase in fluorescence with respect to single fluorochrome bound cells. In another embodiment the first and second detectable markers are different fluorochrome molecules which are selected based on the wavelength at which they fluoresce. The flow cytometric analysis would comprise analyzing the cells at the various wavelengths to determine the presence or absence of both bound fluorochromes.
By xe2x80x9cmultiparameter flow cytometric analysisxe2x80x9d is meant detecting more than one analyte of interest in a sample of cells by flow cytometry.
It is readily recognizable that more than 2 fluorochromes may be selected for the preceding embodiment and a restriction to 4 fluorochromes is presently based on commonly available flow cytometric devices. Hence, at the present up to 4 different molecules may be analyzed by flow cytometric methods. However, flow cytometers are currently available that allow measurement of up to 12 fluorochromes simultaneously, and specialized flow cytometric apparatuses such as are disclosed in U.S. Pat. No. 6,139,800, which is hereby incorporated in its entirety, including all tables, figures, and claims, can be used to increase the number of analytes that may be detected in any one assay. Thus, any improvements to such devices which allow for additional wavelengths or fluorochromes to be distinguished are within the scope of the instant invention. For example, the fluorescent bead technology disclosed in U.S. Pat. No. 5,981,180, which is hereby incorporated in its entirety, including all tables, figures, and claims, may, in principle, allow the detection of thousands of analytes simultaneously.
In preferred embodiments, the present invention provides a method for tyramide labeling of cells for multiple label analysis by flow cytometry. In these methods, multiple binding partners, which can be conjugated to detectable markers, are added to distinguish amongst the analytes of interest. These methods allow for determining the co-expression of markers in and/or on cells.
For example, in double label analysis, cells can be contacted with a first binding partner specific for a first analyte of interest, a second binding partner with enzymatic activity and which specifically binds to the first binding partner, a substrate for the enzymatic activity of the second binding partner, and a labeling molecule containing tyramide. In one embodiment of the present invention, after tyramide deposition and the addition of a detectable marker, the cells are contacted with a third binding partner specific for a second analyte of interest. The third binding partner is preferably conjugated to a detectable marker. In a further embodiment, the third binding partner is added with the addition of the first binding partner. In an even further embodiment of the present invention, the third binding partner is added at anytime during double label analysis as this third binding partner, which is preferably conjugated to a detectable marker, is not directly associated with the amplification of tyramide coating associated with the first and second binding partners.
In another aspect, the present invention discloses materials and methods that provide an enhanced signal for detection of a cell surface analyte of interest in a cell sample by flow cytometric methods. These methods preferably comprise coating cells with tyramide and analyzing the cells with a flow cytometric device.
The terms xe2x80x9ctyramide coatingxe2x80x9d or xe2x80x9ccoating cells with tyramidexe2x80x9d as used herein refer to methods such as those described in U.S. Pat. No. 5,196,306, U.S. Pat. No. 5,583,001, U.S. patent application Ser. No. 09/229,001 and U.S. patent application Ser. No. 09/318,346, each of which is hereby incorporated by reference in its entirety, including all tables, figures, and claims.
In a preferred embodiment, the present invention features a method for tyramide coating cells for flow cytometric analysis by contacting the cells with one or more of the following; a first binding partner specific for the analyte of interest, a second binding partner with enzymatic activity and which specifically binds to the first binding partner, a substrate for the enzymatic activity of the second binding partner, and a labeling molecule containing tyramide. The tyramide-containing labeling molecule is coated on the cells possessing the analyte of interest as a result of the product of the enzymatic activity of the second binding partner and the substrate reacting with the tyramide. A detectable marker may be added after tyramide coating to facilitate flow cytometric analysis. The detectable marker can be a fluorochrome molecule which is attached to a binding partner specific for the tyramide containing molecule. In a preferred embodiment, the tyramide-containing labeling molecule is conjugated to a fluorochrome.
In another embodiment, the cell is transformed to express a molecule that is not a natural component of the cell. These transformed cells may express molecules such as bacterial antigens, viral proteins or cellular proteins normally expressed intracellularly and engineered for expression by the cell. This type of transformation is common and routinely preformed by those in the art and generally involves the insertion of exogenous DNA or RNA constructs composed of a sequence specific for the molecule of interest wherein the construct is configured and arranged in a manner suitable for expression when inside of the cell. Proteins and peptides can also be introduced into cells conjugation to arginine oligomers, HIV-1 tat, or the third helix of Drosophila antennapedia or by complexing with other intracellular delivery vehicles. In addition the analyte of interest can be a molecule which has been inserted into the cell by experimental methods. This molecule may be a dye or a chemical molecule which the cell can internalize or bind on its surface.
In preferred embodiments, the methods for detecting the presence of analytes described herein provide a 5-fold enhancement, a 10-fold enhancement, a 15-fold enhancement, a 20-fold enhancement, a 25-fold enhancement, a 30-fold enhancement, a 40-fold enhancement, a 50-fold enhancement, a 60-fold enhancement, a 75-fold enhancement, a 100-fold enhancement, a 200-fold enhancement, a 500-fold enhancement, and a 1000-fold enhancement in comparison to standard flow cytometry methods.
In an additional embodiment of the invention the binding partner which is specific for the analyte of interest is indirectly bound to an enzyme via another molecule that specifically binds to the binding partner. For example, a binding partner that specifically binds to an analyte of interest can itself be bound by a second antibody that is conjugated to an enzyme. In other embodiments, the binding partner may be conjugated to a molecule such as Fluorescein isothiocyanate (FITC); FITC can then be bound by a second binding partner such as an antibody that specifically binds FITC that is conjugated to an enzyme.
A binding partner can be chemically attached to a hapten, such as FITC, DNP, TNP, digoxigenin, etc., by methods which are routine and well known in the art. In preferred embodiments the binding partner is a haptenated antibody. In a further embodiment the binding partner which is specific for the analyte of interest is a haptenated construct combining a protein or nucleic acid molecule with a hapten such as FITC, DNP, TNP, digoxigenin, etc. Linking the respective binding partners to the hapten molecule prepares the binding partner to be readily available to binding partners which have a high affinity for binding the hapten molecule. Those in the art would readily recognize that other proteins which specifically bind molecules with similar characteristics as FITC and anti-FITC antibodies and which are readily attached to antibodies or cellular analytes are within the scope of the present invention.
In another embodiment the present invention provides a diagnostic method for tyramide coating cells for flow cytometry by removing cells from a patient and contacting the cells with the following, a first binding partner specific for the analyte of interest, a second binding partner with enzymatic activity and which specifically binds to the first binding partner, a substrate for the enzymatic activity of the second binding partner, and a labeling molecule containing tyramide, and a detectable marker.
The term xe2x80x9cdiagnostic methodxe2x80x9d refers to the determination of the nature of a disease. Preferably the disease is caused by a cell, or a changed cell, such as a cancerous cell or a virally infected cell, or a mutated cell, which has a known intracellular or cell surface analyte. Examples of such methods include but are not limited to determining the phenotype of a lymphoma or leukemia, detecting the presence of transformed cells, determining the immunological status of a patient with AIDS or with a primary immunodeficiency syndrome such as severe combined immunodeficiency disease.
In yet another embodiment, the present invention provides an antibody-binding partner conjugate configured and arranged for use with methods for tyramide coating live cells for flow cytometry.
In additional embodiment, the present invention provides a device for flow cytometry comprising tyramide coated cells.
In yet another aspect, the present invention features kits for use with a method of tyramide labeling of analytes for flow cytometry. The kit includes materials for tyramide deposition and/or detecting analytes by flow cytometry. The kit preferably contains components such as, but not limited to, premade buffers, amplification reagents, and a detailed protocol.
In preferred embodiments, the kits of the present invention comprise one or more of the following: an instruction sheet for performing tyramide deposition labeling; a fixation medium; a permeabilization medium; an amplification medium that does not comprise tyramide; fluorescently labeled tyramide in a light-protective container; a primary antibody; a haptenated primary antibody; a secondary anti-primary antibody, which is conjugated to an enzyme such as horseradish peroxidase; a secondary anti-hapten antibody, which is conjugated to an enzyme such as horseradish peroxidase.
In yet another aspect, the present invention features a device for tyramide labeling of analytes for flow cytometry. Preferably such a device is configured and arranged to control the addition of various components required for tyramide deposition as described herein. One of skill in the art would recognize that a device of this manufacture would be configured to incorporate the addition of a sample believed to possess an analyte of interest, the addition of the binding partners of the method, as described above, and would include instrumentation which incorporates intermediate washing steps which are necessary for immunoassays such as flow cytometry, ELISA, radio-immunoassays, analyte dependent enzyme activation system (ADEAS) assays, catalyzed reporter deposition amplification assays, and the like, or other immunohistochemical staining methods.
In yet another aspect, the present invention features compositions for use in catalyzed deposition methods. The compositions can provide reduced nonspecific backgrounds and greater peak separation between histograms obtained from cells stained with control immunoglobulin versus cells stained with immunoglobulin specific for an analyte of interest when used in flow cytometry, as well as in histochemical methods for staining analytes of interest present in thin sections, cell preparations, and other applications known to those of ordinary skill in the art.
In one embodiment, a composition of the instant invention comprises a binding partner that specifically binds to an analyte of interest or that binds to a first binding partner, or a binding partner conjugated to an enzyme, in a medium comprising at least about 10% serum. In particularly preferred embodiments, the medium comprises at least about 10% serum, 20% serum, 30% serum, 40% serum, 50% serum, 60% serum, 70% serum, 80% serum, 90% serum, 95% serum, 99% serum, and 100% serum. The percentages are measured by volume, and the term xe2x80x9caboutxe2x80x9d in this context refers to +/xe2x88x920.5%. A preferred serum is fetal bovine serum. In certain embodiments, the composition comprises a binding partner that specifically binds to an analye of interest, in a medium comprising a protein concentration equivalent to that found in the serum-containing media described above. For example, bovine serum albumin could be present in a medium at a concentration equivalent to a medium comprising 10% serum.
In other preferred embodiments, composition also comprises a detergent, such as CHAPS, cholic acid, deoxycholic acid, digitonin, n-dodecyl-xcex2-D-maltoside, glycodeoxycholic acid, n-lauroylsarcosine, lauryl sulfate, saponin, triton X-100, at a concentration of about 0.1% to about 25%. Most preferably, the composition comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, and 25%. The percentages are measured by volume for those detergents that are a liquid, and by weight for those detergents that are solids, and the term xe2x80x9caboutxe2x80x9d in this context refers to +/xe2x88x920.05%. Particularly preferred is a composition comprising one or more antibodies in a medium comprising at least about 95% serum and about 0.2% saponin.
In another embodiment, a composition of the instant invention comprises tyramide conjugated to a detectable label, in a medium that has a low ionic strength and/or comprises an aprotic solvent, as defined herein.
Particularly preferred is a medium comprising about 5% acetone. Most preferred is a medium that also comprises an enzyme capable of catalyzing the deposition of tyramide, and a substrate for use by an enzyme in producing oxygen radicals necessary for tyramide deposition.
In another embodiment, a composition of the instant invention comprises tyramide conjugated to a detectable label, in a buffering agent, as defined herein. Preferred media comprise, for example, glycylglycine at a concentration of between 2 M to 0.0001 M and at a pH of 7 to 9. Most preferred are glycylglycine buffers of 0.1 M, 0.05 M, 0.05 M, 0.02 M, 0.01 M, 0.005, 0.001, and 0.0001 M, having a pH of 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, and 9. The medium can also comprise one or more salts, such as sodium chloride, potassium chloride, magnesium chloride, etc., at a concentration of from 3 M to 0.001 M, and/or one or more aprotic solvents. In preferred embodiments, the salt(s) are at a concentration of 2.5 M, 2M, 1.5 M, 1.25 M, 1 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.45 M, 0.4 M, 0.35 M, 0.3 M, 0.25 M, 0.2 M, 0.15 M, 0.1 M, and 0.05 M. In a particularly preferred embodiment, the amplification medium comprises 0.02 M glycylglycine, pH 8.0, and 1 M NaCl.
In yet another aspect, the present invention features methods for optimizing compositions for use in catalyzed deposition methods by providing a library of amino acid, dipeptide and/or oligopeptide buffering agents as defined herein. In these methods, a plurality of buffering agents are compared for their ability to reduce nonspecific background staining and provide greater peak separation between histograms obtained from cells stained with control immunoglobulin versus cells stained with immunoglobulin specific for an analyte of interest when used in catalyzed reporter deposition methods. The methods can provide optimal compositions for use in flow cytometry, and in histochemical methods for staining analytes of interest present in thin sections, cell preparations, particles, and other applications known to those of ordinary skill in the art.
The compositions can also comprise one or more salts, such as sodium chloride, potassium chloride, magnesium chloride, etc., at a concentration of from 3 M to 0.001 M, and/or one or more aprotic solvents. In preferred embodiments, the salt(s) are at a concentration of 2.5 M, 2 M, 1.5 M, 1.25 M, 1 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.45 M, 0.4 M, 0.35 M, 0.3 M, 0.25 M, 0.2 M, 0.15 M, 0.1 M, and 0.05 M.
In another embodiment, the present invention features a composition generated by performing any one of the enzymatic amplification staining methods described herein. For example, a preferred composition can comprise a hapten-conjugated first antibody or an antibody fragment, which specifically binds to an analyte of interest; an enzymeconjugated second antibody or antibody fragment; and a precipitate formed by the interaction of the enzyme with its substrate. Alternatively, a preferred composition can comprise an enzyme-conjugated antibody or antibody fragment; and a precipitate formed by the interaction of the enzyme with its substrate. In particularly preferred embodiments, the composition further comprises one or more cells that comprise the antigen of interest. As discussed herein, such a precipitate, e.g., comprising tyramide, can be formed on cells in solution, or on cells affixed to a surface, e.g., a glass slide.
The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the preferred embodiments, as well as from the claims.